Abstract
We have previously reported the isolation of two forms of cytochrome P-450 (P-450) with ω-hydroxylase activities toward prostaglandin A (PGA) and fatty acids, designated as P-450ka-1 and P-450ka-2, from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)phthalate [Kusunose, E. et al. (1989) J. Biochem. 106, 194-196]. In the present work, we have purified and characterized two additional forms of rabbit kidney fatty acid ω-hydroxylase, designated as P-450kc and P-450kd. The purified P-450kc and P-450kd had specific contents of 13 and 16 nmol of P-450/mg of protein, with apparent molecular weights of 52, 000 and 55, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Both the forms showed absorption maxima at 450 nm in the carbon monoxide-difference spectra for their reduced forms. These P-450s efficiently catalyzed the ω- and ω-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and pal-mitate, in a reconstituted system containing P-450, NADPH-P-450 reductase, and phos-phatidylcholine. Cytochrome b5 stimulated the reactions to only a slight extent. They had no detectable activity toward PGA and several xenobiotics tested. The two P-450s showed different peptide map patterns after limited proteolysis with papain or Staphylococcus aureus V8 protease. P-450kd was identical to rabbit pulmonary prostaglandin ω-hydrox-ylase (P-450p-2, P-450IVA4) in its first 20 NH2-terminal amino acid sequence (Ala-Leu-Ser-Pro- Thr-Arg-Leu-Pro-Gly-Ser-Leu-Ser-Gly-Leu-Leu-Gln-Val-Ala-Ala-Leu), although these P-450s were quite different in their other properties such as apparent molecular weights, catalytic activities, inducibility, and tissue distribution. P-450kd and P-450kc were also very similar to P-450ka-1 and P-450ka-2 in their terminal amino acid sequences. The results suggest that all these rabbit renal and pulmonary ω-hydroxylases are members of the same gene family (P-450IVA gene subfamily) in the P-450 superfamily.
